Biomedical Applications of Mid-Infrared Spectroscopic Imaging and Multivariate Data Analysis: Contribution to the Understanding of Diabetes Pathogenesis

Diabetic retinopathy (DR) is a microvascular complication of diabetes and a leading cause of adult vision loss. Although a great deal of progress has been made in ophthalmological examinations and clinical approaches to detect the signs of retinopathy in patients with diabetes, there still remain outstanding questions regarding the molecular and biochemical changes involved. To discover the biochemical mechanisms underlying the development and progression of changes in the retina as a result of diabetes, a more comprehensive understanding of the bio-molecular processes, in individual retinal cells subjected to hyperglycemia, is required. Animal models provide a suitable resource for temporal detection of the underlying pathophysiological and biochemical changes associated with DR, which is not fully attainable in human studies. In the present study, I aimed to determine the nature of diabetes-induced, highly localized biochemical changes in the retinal tissue from Ins2Akita/+ (Akita/+; a model of Type I diabetes) male mice with different duration of diabetes. Employing label-free, spatially resolved Fourier transform infrared (FT-IR) imaging engaged with chemometric tools enabled me to identify temporal-dependent reproducible biomarkers of the diabetic retinal tissue from mice with 6 or 12 weeks, and 6 or 10 months of diabetes. I report, for the first time, the origin of molecular changes in the biochemistry of individual retinal layers with different duration of diabetes. A robust classification between distinctive retinal layers - namely photoreceptor layer (PRL), outer plexiform layer (OPL), inner nuclear layer (INL), and inner plexiform layer (IPL) - and associated temporal-dependent spectral biomarkers, were delineated. Spatially-resolved super resolution chemical images revealed oxidative stress-induced structural and morphological alterations within the nucleus of the photoreceptors. Comparison among the PRL, OPL, INL, and IPL suggested that the photoreceptor layer is the most susceptible layer to the oxidative stress with short-duration of diabetes. Moreover, for the first time, we present the temporal-dependent molecular alterations for the PRL, OPL, INL, and IPL from Akita/+ mice, with progression of diabetes. These findings are potentially important and may be of particular benefit in understanding the molecular and biological activity of retinal cells during oxidative stress in diabetes. Our integrating paradigm provides a new conceptual framework and a significant rationale for a better understanding of the molecular and cellular mechanisms underlying the development and progression of DR. This approach may yield alternative and potentially complimentary methods for the assessment of diabetes changes. It is expected that the conclusions drawn from this work will bridge the gap in our knowledge regarding the biochemical mechanisms of the DR and address some critical needs in the biomedical community

Temporal diabetes-induced biochemical changes in distinctive layers of mouse retina

To discover the mechanisms underlying the progression of diabetic retinopathy (DR), a more comprehensive understanding of the biomolecular processes in individual retinal cells subjected to hyperglycemia is required. Despite extensive studies, the changes in the biochemistry of retinal layers during the development of DR are not well known. In this study, we aimed to determine a more detailed understanding of the natural history of DR in Akita/+ (type 1 diabetes model) male mice with different duration of diabetes. Employing label-free spatially resolved Fourier transform infrared (FTIR) chemical imaging engaged with multivariate analysis enabled us to identify temporal-dependent reproducible biomarkers of the individual retinal layers from mice with 6 weeks,12 weeks, 6 months, and 10 months of age. We report, for the first time, the nature of the biochemical alterations over time in the biochemistry of distinctive retinal layers namely photoreceptor retinal layer (PRL), inner nuclear layer (INL), and plexiform layers (OPL, IPL). Moreover, we present the molecular factors associated with the changes in the protein structure and cellular lipids of retinal layers induced by different duration of diabetes. Our paradigm provides a new conceptual framework for a better understanding of the temporal cellular changes underlying the progression of DR

Quantifying Biochemical alterations in Brown and subcutaneous White adipose Tissues of Mice Using Fourier Transform infrared Widefield imaging

Stimulating increased thermogenic activity in adipose tissue is an important biological target for obesity treatment, and label-free imaging techniques with the potential to quantify stimulation-associated biochemical changes to the adipose tissue are highly sought after. In this study, we used spatially resolved Fourier transform infrared (FTIR) imaging to quantify biochemical changes caused by cold exposure in the brown and subcutaneous white adipose tissues (BAT and s-WAT) of 6 week-old C57BL6 mice exposed to 30°C (N = 5), 24°C (N = 5), and 10°C (N = 5) conditions for 10 days. Fat exposed to colder temperatures demonstrated greater thermogenic activity as indicated by increased messenger RNA expression levels of a panel of thermogenic marker genes including uncoupling protein 1 (UCP-1) and Dio2. Protein to lipid ratio, calculated from the ratio of the integrated area from 1,600 to 1,700 cm−1 (amide I) to the integrated area from 2,830 to 2,980 cm−1 (saturated lipids), was elevated in 10°C BAT and s-WAT compared to 24°C (p = 0.004 and p \u3c 0.0001) and 30°C (p = 0.0033 and p \u3c 0.0001). Greater protein to lipid ratio was associated with greater UCP-1 expression level in the BAT (p = 0.021) and s-WAT (p = 0.032) and greater Dio2 expression in s-WAT (p = 0.033). The degree of unsaturation, calculated from the ratio of the integrated area from 2,992 to 3,020 cm−1 (unsaturated lipids) to the integrated area from 2,830 to 2,980 cm−1 (saturated lipids), showed stepwise decreases going from colder-exposed to warmer-exposed BAT. Complementary 1H NMR measurements confirmed the findings from this ratio in BAT. Principal component analysis applied to FTIR spectra revealed pronounced differences in overall spectral characteristics between 30, 24, and 10°C BAT and s-WAT. Spatially resolved FTIR imaging is a promising technique to quantify cold-induced biochemical changes in BAT and s-WAT in a label-free manner

Estimating and correcting interference fringes in infrared spectra in infrared hyperspectral imaging

Short-term acclimation response of individual cells of Thalassiosira weissflogii was monitored by Synchrotron FTIR imaging over the span of 75 minutes. The cells, collected from batch cultures, were maintained in a constant flow of medium, at an irradiance of 120 μmol m−2 s−1 and at 20 °C. Multiple internal reflections due to the micro fluidic channel were modeled, and showed that fringes are additive sinusoids to the pure absorption of the other components of the system. Preprocessing of the hyperspectral cube (x, y, Abs(λ)) included removing spectral fringe using an EMSC approach. Principal component analysis of the time series of hyperspectral cubes showed macromolecular pool variations (carbohydrates, lipids and DNA/RNA) of less than 2% after fringe correction

Data associated with Retinal oxidative stress at the onset of diabetes determined by synchrotron FTIR widefield imaging: towards diabetes pathogenesis and Temporal diabetes-induced biochemical changes in distinctive layers of mouse retina

This data folder is associated with the following publications in the Analyst journal and the Scientific Reports: 1. Retinal oxidative stress at the onset of diabetes determined by synchrotron FTIR widefield imaging: towards diabetes pathogenesis. (E. Aboualizadeh, et al. February 2017) DOI: 10.1039/C6AN02603F Citation: Aboualizadeh, E., Ranji, M., Sorenson, C. M., Sepehr, R., Sheibani, N., & Hirschmugl, C. J. (2017). Retinal oxidative stress at the onset of diabetes determined by synchrotron FTIR widefield imaging: towards diabetes pathogenesis. Analyst, 142(7), 1061-1072. AND 2. Temporal diabetes-induced biochemical changes in distinctive layers of mouse retina. (E. Aboualizadeh, et al. January 2018) DOI: 10.1038/s41598-018-19425-8 Citation: Aboualizadeh, E., Sorenson, C. M., Schofield, A. J., Unger, M., Sheibani, N., & Hirschmugl, C. J. (2018). Temporal diabetes-induced biochemical changes in distinctive layers of mouse retina. Scientific reports, 8(1), 1096. Abstract of paper 1: Diabetic retinopathy is a microvascular complication of diabetes that can lead to blindness. In the present study, we aimed to determine the nature of diabetes-induced, highly localized biochemical changes in the neuroretina at the onset of diabetes. High-resolution synchrotron Fourier transform infrared (s-FTIR) wide field microscopy coupled with multivariate analysis (PCA–LDA) was employed to identify biomarkers of diabetic retinopathy with spatial resolution at the cellular level. We compared the retinal tissue prepared from 6-week-old Ins2Akita/+ heterozygous (Akita/+, N = 6; a model of diabetes) male mice with the wild-type (control, N = 6) mice. Male Akita/+ mice become diabetic at 4-weeks of age. Significant differences (P \u3c 0.001) in the presence of biomarkers associated with diabetes and segregation of spectra were achieved. Differentiating IR bands attributed to nucleic acids (964, 1051, 1087, 1226 and 1710 cm−1), proteins (1662 and 1608 cm−1) and fatty acids (2854, 2923, 2956 and 3012 cm−1) were observed between the Akita/+ and the WT samples. A comparison between distinctive layers of the retina, namely the photoreceptor retinal layer (PRL), outer plexiform layer (OPL), inner nucleus layer (INL) and inner plexiform layer (IPL) suggested that the photoreceptor layer is the most susceptible layer to oxidative stress in short-term diabetes. Spatially-resolved chemical images indicated heterogeneities and oxidative-stress induced alterations in the diabetic retina tissue morphology compared with the WT retina. In this study, the spectral biomarkers and the spatial biochemical alterations in the diabetic retina and in specific layers were identified for the first time. We believe that the conclusions drawn from these studies will help to bridge the gap in our understanding of the molecular and cellular mechanisms that contribute to the pathobiology of diabetic retinopathy. Abstract of paper 2: To discover the mechanisms underlying the progression of diabetic retinopathy (DR), a more comprehensive understanding of the biomolecular processes in individual retinal cells subjected to hyperglycemia is required. Despite extensive studies, the changes in the biochemistry of retinal layers during the development of DR are not well known. In this study, we aimed to determine a more detailed understanding of the natural history of DR in Akita/+ (type 1 diabetes model) male mice with different duration of diabetes. Employing label-free spatially resolved Fourier transform infrared (FT-IR) chemical imaging engaged with multivariate analysis enabled us to identify temporal-dependent reproducible biomarkers of the individual retinal layers from mice with 6 weeks,12 weeks, 6 months, and 10 months of age. We report, for the first time, the nature of the biochemical alterations over time in the biochemistry of distinctive retinal layers namely photoreceptor retinal layer (PRL), inner nuclear layer (INL), and plexiform layers (OPL, IPL). Moreover, we present the molecular factors associated with the changes in the protein structure and cellular lipids of retinal layers induced by different duration of diabetes. Our paradigm provides a new conceptual framework for a better understanding of the temporal cellular changes underlying the progression of DR. The data folder contains all the data that has been included in this publication. The data files are stored as OPUS files and DPT files. There are files based on the age of mice ranging from 6-weeks to 10 months old diabetic and wild-type mice. The bright field images are also stored in the data folder. The tables contain FT-IR spectra from diabetic and control retina at different duration of diabetes, where each column is one spectrum and the first column in each table shows the wavenumber range. The processed data in this folder are the processed OPUS data in Irydis software and they are saved as PXP file. We included all these data in these 2 mentioned publications. Please feel free to contact either Dr. Carol Hirschmugl ([email protected]) or Dr. Ebrahim Aboualizadeh ([email protected]) for any questions or concerns regarding this data. This second article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder

Opportunities for Live Cell FT-Infrared Imaging: Macromolecule Identification with 2D and 3D Localization

Infrared (IR) spectromicroscopy, or chemical imaging, is an evolving technique that is poised to make significant contributions in the fields of biology and medicine. Recent developments in sources, detectors, measurement techniques and speciman holders have now made diffraction-limited Fourier transform infrared (FTIR) imaging of cellular chemistry in living cells a reality. The availability of bright, broadband IR sources and large area, pixelated detectors facilitate live cell imaging, which requires rapid measurements using non-destructive probes. In this work, we review advances in the field of FTIR spectromicroscopy that have contributed to live-cell two and three-dimensional IR imaging, and discuss several key examples that highlight the utility of this technique for studying the structure and chemistry of living cells

Time lapse synchrotron IR chemical imaging for observing the acclimation of a single algal cell to CO2 treatment

Algae are the main primary producers in aquatic environments and therefore of fundamental importance for the global ecosystem. Mid-infrared (IR) microspectroscopy is a non-invasive tool that allows in principle studying chemical composition on a single-cell level. For a long time, however, mid-infrared (IR) imaging of living algal cells in an aqueous environment has been a challenge due to the strong IR absorption of water. In this study, we employed multi-beam synchrotron radiation to measure time-resolved IR hyperspectral images of individual Thalassiosira weissflogii cells in water in the course of acclimation to an abrupt change of CO2 availability (from 390 to 5000 ppm and vice versa) over 75 minutes. We used a previously developed algorithm to correct sinusoidal interference fringes from IR hyperspectral imaging data. After preprocessing and fringe correction of the hyperspectral data, principal component analysis (PCA) was performed to assess the spatial distribution of organic pools within the algal cells. Through the analysis of 200,000 spectra, we were able to identify compositional modifications associated with CO2 treatment. PCA revealed changes in the carbohydrate pool (1200-950 cm-1), lipids (1740, 2852, 2922 cm-1), and nucleic acid (1160 and 1201 cm-1) as the major response of exposure to elevated CO2 concentrations. Our results show a local metabolism response to this external perturbation

Data associated with Quantifying Biochemical Alterations in Brown and Subcutaneous White Adipose Tissues of Mice Using Fourier Transform Infrared Widefield Imaging

This data folder is associated with the following publication in the Journal of Frontiers in Endocrinology, obesity section: Quantifying Biochemical Alterations in Brown and Subcutaneous White Adipose Tissues of Mice Using Fourier Transform Infrared Widefield Imaging The paper has been published by E. Aboualizadeh, et al. in May 2017. You can access the paper via the following link: https://doi.org/10.3389/fendo.2017.00121 When using the data, please cite the publication. Here is the abstract of the publication: “Stimulating increased thermogenic activity in adipose tissue is an important biological target for obesity treatment, and label-free imaging techniques with the potential to quantify stimulation-associated biochemical changes to the adipose tissue are highly sought after. In this study, we used spatially resolved Fourier transform infrared (FTIR) imaging to quantify biochemical changes caused by cold exposure in the brown and subcutaneous white adipose tissues (BAT and s-WAT) of 6 week-old C57BL6 mice exposed to 30°C (N = 5), 24°C (N = 5), and 10°C (N = 5) conditions for 10 days. Fat exposed to colder temperatures demonstrated greater thermogenic activity as indicated by increased messenger RNA expression levels of a panel of thermogenic marker genes including uncoupling protein 1 (UCP-1) and Dio2. Protein to lipid ratio, calculated from the ratio of the integrated area from 1,600 to 1,700 cm−1 (amide I) to the integrated area from 2,830 to 2,980 cm−1 (saturated lipids), was elevated in 10°C BAT and s-WAT compared to 24°C (p = 0.004 and p \u3c 0.0001) and 30°C (p = 0.0033 and p \u3c 0.0001). Greater protein to lipid ratio was associated with greater UCP-1 expression level in the BAT (p = 0.021) and s-WAT (p = 0.032) and greater Dio2 expression in s-WAT (p = 0.033). The degree of unsaturation, calculated from the ratio of the integrated area from 2,992 to 3,020 cm−1 (unsaturated lipids) to the integrated area from 2,830 to 2,980 cm−1 (saturated lipids), showed stepwise decreases going from colder-exposed to warmer-exposed BAT. Complementary 1H NMR measurements confirmed the findings from this ratio in BAT. Principal component analysis applied to FTIR spectra revealed pronounced differences in overall spectral characteristics between 30, 24, and 10°C BAT and s-WAT. Spatially resolved FTIR imaging is a promising technique to quantify cold-induced biochemical changes in BAT and s-WAT in a label-free manner. “ The data folder contains all the data that has been included in this publication. The data files are stored as OPUS files and DPT files in the order of dates that we collected data. The DPT data are FTIR spectra that were collected via MCT data and the opus data are the hyperspectral data that has been used for showing images in the paper. Files contain FT-IR spectra from 10C, 24C, and 30C brown, subcutaneous, and visceral mouse adipose tissues as well as some ratiometric studies to calculate the protein:lipid ratios in FT-IR spectra in each tissue. Please feel free to contact either Dr. Carol Hirschmugl ([email protected]) or Dr. Ebrahim Aboualizadeh ([email protected]) for any questions or concerns regarding this data

Quantifying Biochemical Alterations in Brown and Subcutaneous White Adipose Tissues of Mice Using Fourier Transform Infrared Widefield Imaging

Stimulating increased thermogenic activity in adipose tissue is an important biological target for obesity treatment, and label-free imaging techniques with the potential to quantify stimulation-associated biochemical changes to the adipose tissue are highly sought after. In this study, we used spatially resolved Fourier transform infrared (FTIR) imaging to quantify biochemical changes caused by cold exposure in the brown and subcutaneous white adipose tissues (BAT and s-WAT) of 6 week-old C57BL6 mice exposed to 30°C (N = 5), 24°C (N = 5), and 10°C (N = 5) conditions for 10 days. Fat exposed to colder temperatures demonstrated greater thermogenic activity as indicated by increased messenger RNA expression levels of a panel of thermogenic marker genes including uncoupling protein 1 (UCP-1) and Dio2. Protein to lipid ratio, calculated from the ratio of the integrated area from 1,600 to 1,700 cm−1 (amide I) to the integrated area from 2,830 to 2,980 cm−1 (saturated lipids), was elevated in 10°C BAT and s-WAT compared to 24°C (p = 0.004 and p < 0.0001) and 30°C (p = 0.0033 and p < 0.0001). Greater protein to lipid ratio was associated with greater UCP-1 expression level in the BAT (p = 0.021) and s-WAT (p = 0.032) and greater Dio2 expression in s-WAT (p = 0.033). The degree of unsaturation, calculated from the ratio of the integrated area from 2,992 to 3,020 cm−1 (unsaturated lipids) to the integrated area from 2,830 to 2,980 cm−1 (saturated lipids), showed stepwise decreases going from colder-exposed to warmer-exposed BAT. Complementary 1H NMR measurements confirmed the findings from this ratio in BAT. Principal component analysis applied to FTIR spectra revealed pronounced differences in overall spectral characteristics between 30, 24, and 10°C BAT and s-WAT. Spatially resolved FTIR imaging is a promising technique to quantify cold-induced biochemical changes in BAT and s-WAT in a label-free manner

Understanding the antimicrobial activity of selected disinfectants against methicillin-resistant Staphylococcus aureus (MRSA).

Disinfectants and biocidal products have been widely used to combat Methicillin-resistant Staphylococcus aureus (MRSA) infections in homes and healthcare environments. Although disruption of cytoplasmic membrane integrity has been documented as the main bactericidal effect of biocides, little is known about the biochemical alterations induced by these chemical agents. In this study, we used Fourier transform infrared (FT-IR) spectroscopy and chemometric tools as an alternative non-destructive technique to determine the bactericidal effects of commonly used disinfectants against MRSA USA-300. FTIR spectroscopy permits a detailed characterization of bacterial reactivity, allowing an understanding of the fundamental mechanism of action involved in the interaction between bacteria and disinfectants. The disinfectants studied were ethanol 70% (N = 5), isopropanol (N = 5), sodium hypochlorite (N = 5), triclosan (N = 5) and triclocarban (N = 5). Results showed less than 5% colony forming units growth of MRSA treated with triclocarban and no growth in the other groups. Nearly 70,000 mid-infrared spectra from the five treatments and the two control (untreated; N = 4) groups of MRSA (bacteria grown in TSB and incubated at 37°C (Control I) / at ambient temperature (Control II), for 24h) were pre-processed and analyzed using principal component analysis followed by linear discriminant analysis (PCA-LDA). Clustering of strains of MRSA belonging to five treatments and the discrimination between each treatment and two control groups in MRSA (untreated) were investigated. PCA-LDA discriminatory frequencies suggested that ethanol-treated spectra are the most similar to isopropanol-treated spectra biochemically. Also reported here are the biochemical alterations in the structure of proteins, lipid membranes, and phosphate groups of MRSA produced by sodium hypochlorite, triclosan, and triclocarban treatments. These findings provide mechanistic information involved in the interaction between MRSA strains and hygiene products; thereby demonstrating the potential of spectroscopic analysis as an objective, robust, and label-free tool for evaluating the macromolecular changes involved in disinfectant-treated MRSA